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PubMed1987

Mycoplasma-induced degradation of IgE bound by Fc epsilon receptors of rat basophilic leukemia cells.

Previous studies from this laboratory have shown that Mycoplasma hyorhinis interacts with some unoccupied, high-affinity 45-kDa receptors for IgE on rat basophilic leukemia (RBL) cells to induce the formation of 71-kDa receptors for IgE. The present study demonstrates that when IgE is bound to the high-affinity receptors, exposure of RBL cells to mycoplasma leads to a time-dependent degradation of the cell-bound IgE into fragments of 186 kDa, 158 kDa and 115 kDa, all of which remain bound to the receptors. Upon reduction, these fragments yield 67-kDa and 55-kDa epsilon chain-derived polypeptides. The degradation appears to start at the N-terminus of the IgE, leading eventually to a complete loss of L chains. In the absence of mycoplasma, the IgE remains relatively intact throughout the same time period with a molecular mass of 210 kDa. The observed degradation of receptor-bound IgE by mycoplasma, should it also occur in vivo, could have important consequences as far as the IgE-dependent mediator release is concerned.

Animals↗

PubMed1991

Functional expression of the endogenous Thy-1 gene and the transfected murine Thy-1.2 gene in rat basophilic leukemia cells.

An interaction of MRC OX7 monoclonal antibody with Thy-1.1 antigen of rat peritoneal and pleural mast cells has been previously shown to induce rat mast cell activation (L. Dráberová, Eur. J. Immunol. 1989, 19: 1715). In the present study we analyzed the expression and function of the Thy-1 antigen in rat basophilic leukemia cells, clone RBL-2H3. Two RBL-2H3-derived cell lines with stable expression of murine Thy-1.2 antigen, after transfection of a genomic clone of murine Thy-1.2 or Thy-1.2 cDNA under the control of simian virus 40 promoter, were also analyzed. Direct radioantibody binding assays, indirect immunofluorescence studies and flow cytometry analyses revealed that both endogenous Thy-1.1 and transfected murine Thy-1.2 gene products were expressed on the surface of the cells under study. Analysis of the distribution of the Thy-1 antigen in situ in cells grown attached to tissue culture vessels located the Thy-1 predominantly in regions of cell-cell contacts. Incubation of RBL-2H3 cells with Thy-1.1-specific antibodies, or of the transfected cells with both Thy-1.1- and Thy-1.2-specific antibodies, induced a rapid early increase in the concentration of intracellular free calcium [( Ca2+]i) released from internal stores. Sustained increase of [Ca2+]i required the presence of Ca2+ in the extracellular medium. The increase in [Ca2+]i was followed by histamine release from the target cells. The combined data indicate that RBL-derived cells can be used as a useful model system for analysis of Thy-1 antigen-mediated activation of rat mast cells.

Animals↗

PubMed1993

Human IgE mediates stimulus secretion coupling in rat basophilic leukemia cells transfected with the alpha chain of the human high-affinity receptor.

Cell surface expression of the alpha chain of the human (h) Fc epsilon receptor type 1 (Fc epsilon R1) can be observed following stable transfection of RBL-2H3 cells with the h alpha chain of the Fc epsilon R1 complex. Association and dissociation constants for hIgE are similar to those previously reported for the ligand and its receptor. The demonstration of mediator release following the cross-linking of hFc epsilon R1 alpha by hIgE and antigen or anti-hFc epsilon R1 alpha antibody suggests that this system will assist the identification of structural motifs and specific amino acids that are involved in the generation of the signal(s) that will initiate and/or propagate the secretion of mediators from mast cells and basophils.

Adenosine↗

PubMed1994

Mutation of aspartate 82 of the human C5a receptor abolishes the secretory response to human C5a in transfected rat basophilic leukemia cells.

C5a is a potent chemoattractant for monocytes, neutrophils and other leukocytes. The receptor for human C5a is a member of the rhodopsin superfamily of G protein-coupled receptors and contains an aspartate residue (Asp82) within the putative second transmembrane domain conserved in all other G protein-linked receptors. We investigated the role of this residue and also the carboxy-terminal 23 residues of the C5a receptor in ligand binding and signal transduction by expressing wild-type and mutant receptors in the rat basophilic leukemia cell line RBL-2H3. Wild-type and truncated receptors coupled efficiently to effector systems, resulting in the C5a-dependent discharge of granule contents. In contrast RBL cells transfected with receptors in which Asp82 had been mutated to asparagine did not respond to human C5a by secretion despite binding human C5a with high affinity. We conclude therefore that Asp82 is not involved in the interaction with ligand but is essential for the proper transduction of the ligand binding signal.

Animals↗

PubMed1995

Thy-1-mediated activation of rat basophilic leukemia cells does not require co-expression of the high-affinity IgE receptor.

The glycosylphosphatidylinositol (GPI)-anchored protein Thy-1 is one of the most abundant molecules expressed on the surface of rat mast cells and rat basophilic leukemia cells, RBL-2H3. Antibody-mediated aggregation of Thy-1 induces in these cells release of secretory components; so does aggregation of the receptor with high affinity for IgE (Fc epsilon RI). To examine whether there is any relationship between Thy-1- and Fc epsilon RI-mediated activation, we have isolated from mutagenized RBL-2H3 cells a variant cell line deficient in the expression of surface Fc epsilon RI, and analyzed its ability to be activated by an antibody to Thy-1. Northern and immuno-blot analyses revealed that the variant cells were deficient in the expression of a structural or a regulatory gene for Fc epsilon RI gamma subunit. The cells did not respond by release of secretagogues and protein-tyrosine phosphorylation to IgE and antigen and anti-Fc epsilon RI monoclonal antibody (mAb) but their response to anti-Thy-1.1 mAb and calcium ionophore A23187 was retained. Transfection of the cloned Fc epsilon RI gamma subunit into the variant cells restored the surface expression of Fc epsilon RI and responsiveness to both the antigen and anti-Fc epsilon RI mAb but had no effect on responsiveness to anti-Thy-1 mAb. The combined data indicate that aggregation of surface Thy-1 glycoproteins activates a metabolic pathway which is independent of the presence of Fc epsilon RI gamma subunit and surface expression of Fc epsilon RI.

Animals↗

PubMed1995

Characterization of Fc gamma receptors on rat mucosal mast cells using a mutant Fc epsilon RI-deficient rat basophilic leukemia line.

A novel rat basophilic leukemia (RBL) 2H3 subline of rat mucosal mast cells deficient in the expression of the gamma chain (RBL-gamma-) has permitted functional characterization of their low-affinity Fc gamma receptors (Fc gamma R). A rat Fc gamma RII analog of the mouse b2 isoform has been earlier identified and its transcript detected in RBL-2H3 cells. We have noew isolated and sequenced the rat Fc gamma RIIb1 isoform and observed differences between its expression in RBL-2H3 and RBL-gamma-. Furthermore, we demonstrate that rat mucosal mast cells express a second, low-affinity Fc gamma receptor, namely the Fc gamma RIII. Stimulation of either cell line with IgG complexes decreased the expression of transcripts for all Fc gamma R. Hence, ligation of Fc gamma R on rat mucosal mast cells apparently regulate their transcription. Selective stimulation through the Fc gamma RII or Fc gamma RII/III systems, respectively, was accomplished by either using RBL-gamma- line or by saturating the Fc epsilon RI on RBL-2H3 with monomeric IgE. RBL-gamma-cells, which do carry Fc gamma RII (but lack Fc epsilon RI and Fc gamma RIII), do not respond to IgG (and IgE) immune complexes as monitored by specific protein tyrosine phosphorylation, degranulation or cytokine secretion. This finding, together with the restoration of the functional phenotype of parental cells upon gamma chain cDNA transfection into RBL-gamma- cells, unequivocally excludes the possible stimulation of rat mucosal mast cells by clustering of their Fc gamma RII. Fc epsilon RI saturation by IgE on parental RBL-2H3 cells completely blocks their response to IgG immune complexes. Thus the Fc gamma R on these cells do not trigger degranulation and this is not due to the absence of Fc gamma RIII as previously suggested. Therefore, co-clustering of Fc gamma RII and Fc gamma RIII on rat mucosal mast cells does not seem to stimulate them. A possible inhibitory role of Fc gamma RII in this process is suggested and discussed.

Animals↗

PubMed1984

Resolution of cellular compartments involved in membrane potential changes accompanying IgE-mediated degranulation of rat basophilic leukemia cells.

The overall membrane potential of rat basophilic leukemia cells (RBL-2H3) calculated from the transmembrane distribution of the lipophilic, tritium-labelled cation tetraphenyl-phosphonium [( 3H]TPP+) was resolved into its mitochondrial and plasma membrane potential components. Using the mitochondrial uncoupler carbonylcyanide-p-trifluormethoxyphenyl hydrazone (FCCP) which collapses the mitochondrial potential, it was shown that about one third of the overall potential resulted from the mitochondrial contribution. Degranulation of the RBL cells induced by two different IgE-cross-linking agents (specific antigen and anti-IgE antibodies), was accompanied by, and well correlated with, a decrease in the overall potential. However, evaluation of the source of these observed potential changes revealed that the FCCP-insensitive fraction of the overall potential, delta psi P, (representing the plasma membrane potential), was not affected. In contrast, the FCCP-sensitive component due to the mitochondrial potential decreased when receptor cross-linking increased. Thus, the observed decrease in the overall potential is most probably a secondary event in the sequence leading from stimulus to secretion. Indeed, exposure of the RBL cells either to a high external concentration of K+ ions or to a high amount of external TPP+, both causing depolarization, failed to trigger degranulation. It is suggested that the apparent decrease in the measured overall potential is a reflection of the mitochondrial membrane depolarization. The latter is most probably caused by mitochondrial Ca2+ uptake initiated by the increase in the intracellular concentration of Ca2+ which follows cells activation.

Animals↗

PubMed2003

Induction of leukotriene C4 synthase after the differentiation of rat basophilic leukemia cells with retinoic acid and a low dose of actinomycin D and its suppression with methylprednisolone.

Leukotriene C(4) synthase (LTC(4) S) is a pivotal enzyme for generation of cysteinyl-leukotrienes (cysLTs). LTC(4) S activity in rat basophilic leukemia-1 (RBL-1) cells increased after culture in the presence of retinoic acid (RA) analogues, which was inhibited by cycloheximide or actinomycin D (ACD). Unexpectedly, the co-addition of a low dose of ACD with RA further potentiated the upregulation of the LTC(4) S activity. Daunorubicin and mitomycin C also had a similar effect. When stimulated with calcium ionophore A23187, control cells did not produce cysLTs, but RA-treated cells generated cysLTs and the co-addition of ACD further increased. While LTC(4) S mRNA and protein increased in the cells treated with RA, the co-addition of ACD further potentiated both in proportion to the LTC(4) S activity. The effect of ACD was considered to enhance the transcription rate of LTC(4) S gene, but not the mRNA-stability. The addition of methylprednisolone (MP) inhibited generation of cysLTs from the cells with A23187-stimulation and also did LTC(4) S activity, but did not inhibit 5-lipoxygenase (5-LOX). The suppression of LTC(4) S with MP showed a dependent manner on the time-point and duration of MP-treatment after RA-addition which was correlated with reduction in LTC(4) S mRNA and protein. The cells cultured with RA plus ACD contained more histamine, chymase activity, and granules in the cytoplasm than the control cells, suggesting differentiation to mature mast cells. In consideration of RA-differentiation therapy, it may be of pathophysiological relevance that the antineoplastic agents potentiate RA-induced, steroid-sensitive, induction of LTC(4) S in RBL-1 cells.

Animals↗

PubMed1990

Release of calcium from intracellular stores in rat basophilic leukemia cells monitored with the fluorescent probe chlortetracycline.

Release of calcium from intracellular stores of rat basophilic leukemia cells was monitored using the fluorescent probe chlortetracycline. The ability of chlortetracycline to indicate release from intracellular calcium stores was initially validated. The decrease of chlortetracycline fluorescence upon antigen-stimulation was not the result of secretion of granule-associated dye or of changes in the properties of the membranes. The chlortetracycline fluorescence signal was not influenced by Ca2+ influx across the plasma membrane. Results obtained from these chlortetracycline fluorescence measurements corresponded well with 45Ca efflux data, an indirect measurement of release of calcium from stores. Chlortetracycline was used to examine the rate of antigen-induced release of calcium from stores, the depletion of intracellular calcium stores by EGTA, and the relationship between the antigen-stimulated release of stored calcium and exocytosis. Chlortetracycline was shown to be a useful qualitative indicator for the release of intracellular calcium with a relatively rapid response time.

Animals↗

PubMed1992

The inward rectifier potassium conductance in rat basophilic leukemia cells.

Whole-cell recordings were made from rat basophilic leukemia cells. The properties of the cells in the potential range negative to -20 mV could be accounted for by two potassium conductances, a leakage conductance (0.54 +/- 0.01 nS, mean +/- s.e.m., n = 6) and an inward rectifier conductance. The inward rectifier conductance activated at a potential close to EK and had a maximal value of 2.7 +/- 0.22 nS (n = 6). Cesium (2 mM, n = 6) decreased the inward current at every potential. Strontium (10 mM, n = 7) and rubidium (2 mM, n = 3) had a similar effect, but they also increased the slope conductance between -63 and -103 mV. Barium (1-100 microM) blocked selectively the inward rectifying current without affecting the leakage current; the effect was use-, voltage-, and temperature-dependent. TEA decreased the current; the concentration giving 50% inhibition was 37.5 mM. The current was unaffected by phorbol esters as well as several hormones and transmitters that were tested.

Animals↗

PubMed2002

Real-time monitoring of histamine released from rat basophilic leukemia (RBL-2H3) cells with a histamine microsensor using recombinant histamine oxidase.

To detect low levels of histamine, we developed a histamine microsensor using recombinant histamine oxidase. Histamine oxidase with a histidine tag was readily purified using a histidine affinity column. The enzyme showed higher catalytic activity on histamine than diamines (e.g., putrescine and cadaverine) or N(tau)-methylhistamine. The sensor had three carbon film electrodes modified with osmium-polyvinylpyridine-based gel containing horseradish peroxidase, histamine oxidase, and Ag. When a standard solution of histamine was aspirated at a flow rate of 2 microl/min, the detected current was proportional to the histamine concentration and the lower detection limit was 11.3 nM. When rat basophilic leukemia cells (1 x 10(6)) were stimulated by various concentrations of antigen (2, 20, and 200 ng/ml), the histamine concentrations were 0.32, 2.7, and 1.3 microM, respectively, and 20 ng/ml of antigen was found to be the optimal concentration for the antigen-antibody reaction. In contrast, when thapsigargin, an inhibitor of Ca-ATPase in the endoplasmic reticulum, was added (50, 100, and 500 nM), the detected current increased with thapsigargin concentrations and the measured histamine concentrations were 28 nM, 1.3 microM, and 2.7 microM, respectively. These results indicate that the microsensor is useful for the analysis of histamine release from mast cells.

Amine Oxidase (Copper-Containing)↗

PubMed1994

Potent activation of phospholipase D by phenylarsine oxide in rat basophilic leukemia (RBL-2H3) cells.

A putative protein tyrosine phosphatase inhibitor, phenylarsine oxide (PAO), potentiated phospholipase D (PLD) activity concentration-dependently in [3H] oleic acid-labeled rat basophilic leukemia (RBL-2H3) cells without significant increase in phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Although PAO induced tyrosine phosphorylation of several proteins, both PAO-induced PLD activation and tyrosine phosphorylation were not affected by a protein tyrosine kinase inhibitor, genistein. Another tyrosine kinase inhibitor, herbimycin A, prevented the PAO-induced PLD stimulation but had no effect on protein tyrosine phosphorylation. However, depletion of protein kinase C (PKC) greatly reduced PAO-stimulated PLD activity. These results indicate that PKC but not tyrosine kinase may be involved in PAO-mediated PLD activation.

Animals↗

PubMed1994

Cloning of the cDNA for the deleted syk kinase homologous to ZAP-70 from human basophilic leukemia cell line (KU812).

Syk kinase is one of the protein tyrosine kinases and forms a family with ZAP-70. We isolated two different sized cDNA clones of syk (Syk11 and Syk41) from a cDNA library of human KU812 (human basophilic leukemia cell line). The obtained two clones carried different messages, that Syk41 had a 69 bp-long insertion between the SH2 domain and the kinase domain compared with Syk11. Alignment of the two human syk predicted polypeptides with those of the porcine syk and ZAP-70 revealed that human syk was 5 amino acid longer than the porcine syk at the N-termini and that the insertion of the 23 amino acid found in Syk41 was present in the porcine syk and absent in ZAP-70. Reverse transcribed polymerase chain reaction targeting this region showed that both forms of the polyA RNA were expressed in Jurkat cells, human peripheral leukocytes and also KU812 cells and that the inserted form was dominant.

Amino Acid Sequence↗

PubMed1995

Development of a rapid approach to identification of tyrosine phosphorylation sites: application to PKC delta phosphorylated upon activation of the high affinity receptor for IgE in rat basophilic leukemia cells.

In rat basophilic leukemia cells (RBL-2H3) activation of the high affinity receptor for IgE induces tyrosine phosphorylation of PKC delta. We carried out solid phase synthesis of 15 amino acid long oligopeptides corresponding to the sequences around each of the 19 tyrosine residues in PKC delta. Only three oligopeptides, corresponding to tyrosine 52, 155, and 565, were phosphorylated when exposed to lyn kinase. Single mutants in each of these three tyrosine residues of PKC delta were prepared. Upon expression in the RBL-2H3 cells, only the mutant in tryosine 52 showed abolition of the IgE-antigen induced tyrosine phosphorylation.

Amino Acid Sequence↗

PubMed1996

Effect of Clostridium difficile toxin B on IgE receptor-mediated signal transduction in rat basophilic leukemia cells: inhibition of phospholipase D activation.

Antigen (Ag)-stimulated phospholipase D (PLD) activation and secretion were almost abolished by pretreatment of rat basophilic leukemia (RBL)-2H3 cells for 4 h with 5 ng/ml Clostridium difficile Toxin B which is known to inhibit Rho family proteins (Rho, Cdc42, Rac). The concentration-dependent inhibition of PLD activation was well correlated with the level of glucosylation of Rho family proteins. In streptolysin O-permeabilized RBL cells, Toxin B suppressed [3H] phosphatidylbutanol (PBut) formation in response to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and phorbol 12-myristate 13-acetate (PMA) by 67 and 43%, respectively. The synergistic PLD activation by GTP gamma S and PMA was also reduced by Toxin B by 67%. These results suggest that the IgE receptor-coupled PLD activation is largely mediated by Rho proteins.

Animals↗

PubMed1997

Novel inhibitors of brain, neuronal, and basophilic anandamide amidohydrolase.

Mammalian brain as well as mouse neuroblastoma (N18TG2) and rat basophilic leukaemia (RBL) cells were previously shown to contain "anandamide amidohydrolase', a membrane-bound enzyme sensitive to serine and cysteine protease inhibitors and catalyzing the hydrolysis of the endogenous cannabimimetic metabolite, anandamide (arachidonoyl-ethanolamide). With the aim of developing novel inhibitors of this enzyme, we synthesized three arachidonic acid (AA) analogues, i.e. arachidonoyl-diazo-methyl-ketone (ADMK), ara-chidonoyl-chloro-methyl-ketone (ACMK) and O-acetyl-arachidonoyl-hydroxamate (AcAHA), by adding to the fatty acid moiety three functional groups previously used to synthesize irreversible inhibitors of serine and cysteine proteases. The three compounds were purified and characterized by proton nuclear magnetic resonance and electron impact mass spectrometry. Their effect was tested on anandamide amidohydrolase partially purified from N18TG2 and RBL-1 cells and porcine brain. Pre-treatment of the enzyme with each compound produced a significant inhibition, with ADMK being the most potent (IC50 = 3, 2 and 6 microM) and AcAHA the weakest (IC50 = 34, 15 and 25 microM) inhibitors. The inactivated enzyme regained its full activity when chromatographed by anion-exchange chromatography, suggesting that none of the compounds inhibited the amidohydrolase in a covalent manner. Accordingly, Lineweaver-Burk profiles showed competitive inhibition by each compound. Conversely, the irreversible inhibitor of cytosolic phospholipase As, methyl-arachidonoyl-fluoro-phosphonate (MAFP), covalently inhibited the amidohydrolase. MAFP was active at concentrations 10(3) times lower than those reported for phospholipase A2 inhibition, and is the most potent anandamide amidohydrolase inhibitor so far described (IC50 = 1-3 nM). MAFP, ADMK and ACMK, probably by inhibiting anandamide degradation, produced an apparent increase of the in vitro formation of anandamide from its biosynthetic precursor N-arachidonoyl-phosphatidyl-ethanolamine.

Amidohydrolases↗

PubMed2000

Epigallocatechin gallate inhibits histamine release from rat basophilic leukemia (RBL-2H3) cells: role of tyrosine phosphorylation pathway.

Some tea polyphenolic compounds including (-)-epigallocatechin gallate (EGCG) have been shown to inhibit histamine release from mast cells through poorly understood mechanisms. By using a mast cell model rat basophilic leukemia (RBL-2H3) cells we explored the mechanism of the inhibition. EGCG inhibited histamine release from RBL-2H3 cells in response to antigen or the calcium-ionophore A23187, while (-)-epicatechin (EC) had little effect. Increased tyrosine phosphorylation of several proteins including approximately 120 kDa proteins occurred in parallel with the secretion induced by either stimulation. EGCG also inhibited tyrosine phosphorylation of the approximately 120-kDa proteins induced by either stimulation, whereas EC did not. The tyrosine kinase-specific inhibitor piceatannol inhibited the secretion and tyrosine phosphorylation of these proteins induced by either stimulation also. Further analysis showed that the focal adhesion kinase pp125(FAK) was one of the approximately 120-kDa proteins. These findings suggest that EGCG prevents histamine release from mast cells mainly by inhibiting tyrosine phosphorylation of proteins including pp125(FAK).

Animals↗

PubMed1993

Variants of vasoactive intestinal peptide in mouse mast cells and rat basophilic leukemia cells.

Radioimmunoassays for neuroendocrine vasoactive intestinal peptide (VIP1-28) detected 30-120 fmol of structurally related peptides in extracts of 10(7) mouse peritoneal mast cells, bone marrow-derived mast cells, cultured PT-18 and C1.MC/C57.1 lines of mast cells, and rat basophilic leukemia (RBL) cells. No VIP was found in peritoneal cells of mast cell-deficient WBB6F1-W/Wv mice, whereas the amounts extracted from peritoneal cells of the congenic normal (WBB6F1-+/+) mice were similar to those from cultured mouse mast cells. Sephadex G-25 gel filtration resolved two different-sized variants of VIP from mouse mast cells and RBL cells. Amino acid sequence analyses showed that the smaller variant is VIP10-28. The principal amino-terminally larger variant of VIP from C1.MC/C57.1 mouse mast cells and RBL cells exhibited amino acid sequence homology with VIP(-6)-28, and this sequence was established for the corresponding larger VIP from PT-18 mast cells. Polymerase chain reaction amplification of two different substituent sequences of prepro VIP in RBL cell RNA identified the VIP message. VIP10-28 was released from mouse mast cells concurrently with histamine by IgE-dependent stimulation. Rodent mast cell-derived VIP thus consists of both the truncated VIP10-28 and amino-terminally larger forms that appear to be generated by peptidolysis of a preproVIP similar to that found in neural cells.

Amino Acid Sequence↗